Ribosome profiling of selenoproteins in vivo reveals consequences of pathogenic Secisbp2 missense mutations

Abstract

Recoding of UGA codons as selenocysteine (Sec) codons in selenoproteins depends on a selenocysteine insertion sequence (SECIS) in the 3-UTR of mRNAs of eukaryotic selenoproteins. SECIS-binding protein 2 (SECISBP2) increases the efficiency of this process. Pathogenic mutations in SECISBP2 reduce selenoprotein expression and lead to phenotypes associated with the reduction of deiodinase activities and selenoprotein N expression in humans. Two functions have been ascribed to SECISBP2: binding of SECIS elements in selenoprotein mRNAs and facilitation of co-translational Sec insertion. To separately probe both functions, we established here two mouse models carrying two pathogenic missense mutations in Secisbp2 previously identified in patients. We found that the C696R substitution in the RNA-binding domain abrogates SECIS binding and does not support selenoprotein translation above the level of a complete Secisbp2 null mutation. The R543Q missense substitution located in the selenocysteine insertion domain resulted in residual activity and caused reduced selenoprotein translation, as demonstrated by ribosomal profiling to determine the impact on UGA recoding in individual selenoproteins. We found, however, that the R543Q variant is thermally unstable in vitro and completely degraded in the mouse liver in vivo, while being partially functional in the brain. The moderate impairment of selenoprotein expression in neurons led to astrogliosis and transcriptional induction of genes associated with immune responses. We conclude that differential SECISBP2 protein stability in individual cell types may dictate clinical phenotypes to a much greater extent than molecular interactions involving a mutated amino acid in SECISBP2. This work was supported by Deutsche Forschungsgemeinschaft Grants Schw914/2 and Schw914/5 and Universitätsklinikum Bonn. The authors declare that they have no conflicts of interest with the contents of this article. This article was selected as one of our Editors' Picks. This article contains Table S1 and Figs. S1-S3. Raw and processed original data have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) under accession number GSE119681. 1 Supported by a BONFOR SciMed scholarship of the medical faculty.

The Neurobiology of Selenium: Looking Back and to the Future

“…A recent proteome paper now suggested there are additional Sec-containing proteins with UGA/Sec codons, but lacking recognizable SECIS elements ( Guo et al, 2018 ). This provocative finding is, interestingly, in line with the demonstration of selenoprotein translation in the absence of functional SECISBP2 ( Fradejas-Villar et al, 2017 ; Zhao et al, 2019 ). If mutations in SEPSECS , unlike mutations in SECISBP2 , would also affect selenoproteins that do not depend on a SECIS for biosynthesis, the dichotomy of phenotypes could be explained and some of the non-canonical selenoproteins would likely be important for the brain.…”

“…Yet, in humans, mutations most often come as missense, splicing or other mutations that may not completely abrogate 100% of gene/protein expression/activity. We have recently shown that the effect of a missense mutation in vitro and in vivo may differ, in particular stability of a mutated protein may depend on the cell type ( Zhao et al, 2019 ). For the SECISBP2 R 543 Q mutant, we demonstrated that the protein is a complete NULL in mouse liver, but partially functioning and supporting selenoprotein expression in neurons ( Zhao et al, 2019 ).…”

Selenium, Selenoproteins and Viral Infection

“…The selenocysteine insertion sequence (SECIS) located in the 3′ UTR of the mRNA [72] and the selenocysteine-tRNA (Sec-tRNA [Ser]Sec ) [16], together with their interacting protein partners allow the co-translational incorporation of a selenocysteine amino acid in selenoproteins. This mechanism is rather inefficient (between 1 and 5%), and mostly results in a truncated protein, the UGA codon being read as a stop codon [73,74,75,76,77,78]. Interestingly, more and more reports support the idea that the UGA-selenocysteine recoding event by the ribosome is a limiting stage, and its efficiency dictates selenoprotein expression [15,17,22,67,68,69,70,74].…”

Human Disorders Affecting the Selenocysteine Incorporation Pathway Cause Systemic Selenoprotein Deficiency

“…Three missense SECISBP2 mutations (R540Q, C691R and E679D) have been described: The R540Q mutation localises to the K-rich region within the RBD (Figure 2), with the R540Q mutant exhibiting reduced binding to SECIS elements in GPX1 and DIO2 mRNAs, correlating with diminished GPX1 and DIO2 enzyme activity in patient-derived primary cells and mouse model. Detailed analyses suggests that R540Q mutant SECISBP2 fails to bind only a subset of SECIS-elements, consistent with the K-rich region mediating recognition of specific (type I and type II) SECIS elements ( 14) and a mouse model revealed a possible tissue specific pattern of SECISBP2 protein stability correlating with varying loss or preservation of expression of different selenoproteins (14,31,96).…”

“…showing enhanced proteasomal degradation of the C691R mutant SECISBP2 protein, confirmed this (75). A mouse model suggests that the C691R mutant SECISBP2 is unable to bind RNA and non-functional (96). The E679D SECISBP2 mutation is predicted to be deleterious (PolyPhen-2 algorithm score of 0.998 and also located in the RBD and may therefore affect its RNA binding function, but this has not been investigated in detail (37)…”